WO2010134992A2 - Synthèse de désoxyribonucléosides ou de ribonucléosides modifiés par une base labile protégée, phosphoramidites et supports correspondants et leur utilisation dans la synthèse d'oligonucléotides de grande pureté - Google Patents
Synthèse de désoxyribonucléosides ou de ribonucléosides modifiés par une base labile protégée, phosphoramidites et supports correspondants et leur utilisation dans la synthèse d'oligonucléotides de grande pureté Download PDFInfo
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- 0 C*C(*(C(C)C)C(C)C)OC(C1)[C@@](CO)O[C@]1*(C=CC(*[N+]([O-])ONC1c2ccccc2-c2c1cccc2)=*1)C1=O Chemical compound C*C(*(C(C)C)C(C)C)OC(C1)[C@@](CO)O[C@]1*(C=CC(*[N+]([O-])ONC1c2ccccc2-c2c1cccc2)=*1)C1=O 0.000 description 2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- This invention relates to synthesis of novel N-2-acetyl protected deoxy and ribo- guanosine, - N-acetyl protected modified guanosine, their succinates, phosphoramidites, corresponding solid supports that are suitable for high purity DNA and RNA synthesis, and it leads to a novel approach to obtain highest purity oligonucleotides.
- This invention is directed to the synthesis of high purity large scale oligonucleotide synthesis.
- FMOC group was reported for the protection of amino function of 2'- deoxycytidine, 2'-deoxy adenosine and T- deoxy guanosine and for the corresponding ribonucleosides (H. Heikkila and J. Chattopadhyaya, Acta Chem. Scand. B 37, No.3, 263-265, 1983).
- FMOC as n- protecting group as pointed out by these authors for their capability to get cleaved under very mild alkaline deprotection condition, or by bases capable to carry out selective deprotection via B- elimination of FMOC group (scheme 1). It is therefore not surprising that other attempts to synthesis of N- FMOC protected nucleoside and phosphoramidites have been carried out.
- the FMOC protecting group is very well established in peptide synthesis and one of the preferred reagents for amino group protection of alpha- amino group of amino acids for step wise peptide synthesis ( Carpino, L.A., and Han, G. Y., J.Amer.Chem.Soc, 92, 5748, 1970). But Heikkila and Chattopadyaya (J. Heikkila and J. Chattopadhyaya, Acta Chem. Scand. B 37, No.3, 263-265, 1983 ), who initially synthesized the FMOC deoxy and ribo nucleosides switched to ( J. Heikkila, N. Balgobin and J.
- Nps 2- nitrophenyl sulfenyl
- This process therefore offers very attractive potential to use ammonia free oligo synthesis.
- This process furthermore, has potential to offer deoxyoligonucleotides for complete deprotection of oligos on solid supports.
- This technology or process has the potential to offer ribonucleotides such as those required for chip based techniology as well high purity oligonucleotides for microRNA, Si RNA, RNA chips
- This group in conjunction with cyanoethyl phosphate protecting group, therefore offers opportunity to remove both FMOC and cyanoethyl groups from the synthesized deoxy and ribo oligonucleotides on the support cleanly, preferably with non aq bases, and on support for many diagnostics application.
- Our present invention is revealed by structures such as 7-10 below.
- the present inventors recently proposed to utilize N- FMOC protected purine and pyrimidine bases for the Sythesis of protected Deoxy nucleosides, ribonucleosides, phosphoromidites and their use in the synthesis of oligonucleotides.
- the FMOC protecting group presents tremendous advantage, practical large scale synthesis is inconvenient and difficult to achieve in order to produce large quantities of therapeutic grade RNA or RNA chimeras having sensitive groups such as 2'- fluoro group. It is also significantly labile so that this group has a tendency to fall off and lead to byproducts or impurities. We therefore turned our attention to N-2- acetyl as guanine base protecting group. This group is significantly more stable than FMOC protecting group. Yet is quite labile as compared to isobutyryl group at N-2 position.
- N-2 acetyl guanine in a nucleoside or oligonucleotide is significantly faster as compared to standard N-2 isobutyryl group on guanine residue, so that the quality of oligonucleotides especially RNA and modified RNA increases dramatically.
- oligo ribonucleotide chimeras comprise of mixed bases composed of 2'- fluoro and T- ribo bases they present a challenge in obtaining pure chimera oligonucleotides. It has been well documented by several recent reports that oligonucleotide chimeras having 2'-fluoro-2;'-deoxy bases along with natural ribo bases present difficulty in obtaining pure oligos. It has been shown that with strongly basic conditions, there is significant loss of fluorine as loss of Hydrogen Fluoride (“HF”) is seen as M-20 peak in Mass spectral analysis.
- HF Hydrogen Fluoride
- T- fluoro-2'- deoxy nucleosides and corresponding phosphoramidites with guanine protecting groups having N-2 acetyl guanine for mild and shorter base deprotection protocol.
- N-2- acetyl guanosine protecting group still high purity and integrity oligo chimeras is obtained and RNA high quality for therapeutic and diagnostic applications, such as for applications in SiRNA synthesis.
- 2'-O-alkyl nucleoside phosphoramidites are extensively used in the design of biologically active oligonuceotides for therapeutic and diagnostic applications as fully alkylated or as chimeras.
- 2'-O-alkyl nucleosides and phosphoramidites the most common are 2'-O-Methyl oligonucleotides which have shown enormous promise in drug design and specific diagnostics applications.
- T- Omethyl oligoribonucleotides- RNA complexes have higher Tm than corresponding oligo-deoxy ribonucleoside - RNA duplexes, Iribarren, A.M., Sproat, B. S., Neuner, P., Sulston, I., Ryder, U., and Lamond, A.I., Proc. Natl. Acad. Sci. USA 87, 7747-7751, 1990.
- Various T- OMethyl-N-FMOC protected nucleosides and phosphoramidites offer great advantage to produce high quality of DNA- RNA oligonucleotides and chimera for biological applications.
- RNA synthesis is now well established and currently in use for synthesis and development of vast variety of therapeutic grade RNA aptamers, tRNA's, Si RNA and biologically active RNA molecules.
- This approach utilizes a ribonucleoside with suitable N- protecting group, 5'- Protecting group, generally, and most popular being dimethoxytriphenyl, commonly called DMT group, T- protecting group, out of which most popular is t-Butyldimethylsilyl ether and a 3'- phosphoramidite, wherein the most popular still is cyanoethyl diisopropyl (component 1).
- This component is then coupled with a nucleoside with a suitable N- protecting group, 2' or 3' succinate of a ribonucleoside attached to a solid support.
- the coupling of component 1 and 5'- OH-n-protected -2',3'-protected- nucleoside are also achieved in solution phase in the presence of an activator to lead to dimers and oligoribonucleotides, followed by oxidation ( 3'->5' direction synthesis), also lead to protected dinucleoside having a 3 '-5'- internucleotide linkage, Ogilvie, K.K., Can. J. Chem., 58, 2686, 1980.
- N- acetyl guanine protecting group offers great potential in RNA synthesis of defined sequence based on our invention as outlined here.
- T- protecting group can be utilized in conjunction with various T- protecting groups required for RNA synthesis.
- T- protecting group tert- butyl-dimethylsilyl
- DTM t- butyldithiomethyl
- T- protecting group acetal levulinyl ester (ALE)
- structure 15 has been recently proposed ( J.G. Lackey and M.J.Damha, Nucleic Acids Symposium Series, No. 52, 35-36, 2008).
- T- O-acetal ester, pivaloyloxy methyl which has been found mild T- O protercting group,T. Lavergne, A. Martin, F. Debart, J-J Vasseur, Nucleic Acids Symposium Series No. 52, , 51-52, 2008.
- the base protecting group used by these authors was n- acetyl and tbPAC. This gives additional credence to our process.
- n- acetyl for RNA synthesis with other T- protecting groups or T- modification would be an ideal group for deprotection under mild basic condition in shorter period of time. '
- RNA Chemically modified RNA have been synthesized having modified arabino sugars, 2'-deoxy- T- fluoro- beta-D_arabinonucleic acid ( FANA; structure 17)) and 2'-deoxy-4'- thio-2'- fluoro- beta-D arabinonucleic acid (4'-Thio-FANA; structure 18) into sequences for SiRNA activities, Dowler, T., Bergeron, D., Tedeschi, Anna-Lisa, Paquet, L., Ferrari, N., Damha, M.J., Nucl. Acids Res., 34, 1669-1675, 2006.
- T- protecting 2-cyanoethoxymethyl (CEM) structure 19
- CEM 2-cyanoethoxymethyl
- N- acetyl protected guanine as nucleoside base having the T- protecting group discussed above can be combined and utilized in high purity RNA synthesis.
- the N- acetyl guanine protected nucleoside offers great potential in RNA synthesis of defined sequence. This applies to RNA synthesis in the conventional direction (3' -> 5' direction as well as using our newly discovered 5'-> 3' direction synthons; structures 21, 22 & 23). Structures of reverse phosphoramidites and solid supports:
- Chemical synthesis of RNA is desirable because it avoids the inefficiencies and limitation of scale of synthesis such as by in vitro transcription by T7 RNA polymerase, Helm, M., Brule, H., Giege, R., Florence, C, RNA, 5:618-621, 1999.
- RNA tertiary structures Chemical synthesis of RNA is desirable for studies of RNA structure and function, and many useful modifications can be achieved selectively, such as site specific introduction of functional groups;viz., disulphide cross linking as a probe of RNA tertiary structures, Maglott, E.J., Glick, G. D., Nucl. Acids Res., 26: 1301-1308, 1999.
- Synthesis of long RNA is very important for biologically active molecules such as tRNA, and such synthesis have been achieved, Persson, T., Kutzke, U., Busch, S., Held, R., Harmann, R.K., Bioorgan. Med.
- This invention relates to novel synthesis methodology utilizing N- acetyl guanine protected nucleosides, succinates, phosphoramidites, corresponding solid supports that are suitable for oligo deoxy nucleosides and RNA oligonucleotide synthesis.
- N- acetyl as nucleoside base protecting group, which is more base labile protecting group as compared to n- isobutyryl group is a novel approach to obtain highest purity oligonucleotides.
- This approach is designed to lead to very high purity and very clean oligonucleotide, after removal of the protecting groups and efficiently in order to produce high purity and therapeutic grade DNA oligonucleotides, RNA oligonucleotides, diagnostic DNA, diagnostic RNA for microarray platform.
- the deprotection of acetyl protecting groups of the natural deoxy and ribonucleosides occurs under the mild deprotection and shorter contact time deprotection conditions and removal of such groups under very mild conditions would allows synthesis of various DNA and RNA of highest purity for diagnostics and therapeutic application.
- This approach is further designed to use acetyl guanine protecting group on various base sensitive nucleoside, and for use in oligo peptide synthesis.
- RNA synthesis is now well established and currently in use for synthesis and development of vast variety of therapeutic grade RNA aptamers, tRNA's, Si RNA and biologically active RNA molecules.
- This approach utilizes a ribonucleoside with suitable N- protecting group, 5'- Protecting group, generally and most popular being dimethoxytriphenyl, commonly called DMT group, T- protecting group, out of which most popular being t-Butyldimethylsilyl ether and a 3'- phosphoramidite, the most popular being cyanoethyl diisopropyl (component 1).
- This component is then coupled with a nucleoside with a suitable N- protecting group, T or 3' succinate of a ribonucleoside attached to a solid support (component T).
- component 3 The coupling of component 1 and 5'- OH-n- protected -2',3'-protected-nucleoside ( component 3) are also achieved in solution phase in presence of an activator to lead to dimers and oligoribonucleotides, followed by oxidation ( 3'->5' direction synthesis), also lead to protected dinucleoside having a 3'-5'- internucleotide linkage, Ogilvie, K.K., Can. J. Chem., 58, 2686, 1980.
- TBDMS group which has been extensively developed by Ogilvie and coworkers as T- hydroxy protecting group for oligo ribonucleotide synthesis
- DTM t- butyldithiomethyl
- RNA interference RNA interference
- RNA interference in Caenorhabditis elegans is mediated by 21 and 22 nucleotide RNA sequences. This was further confirmed as general phenomenon of specific inhibition of gene expression by small double stranded RNA's being mediated by 21 and 22 nucleotide RNA's, Genes Dev., 15, 188-200, 2001.
- the invention provides a novel method of RNA synthesis utilizing N-2- acetyl guanosine- 5'- DMT-2' -TBDMS- protected nucleoside phosphoramidite and other nucleoside phosphoramidites outlined in Formula 1 below.
- the synthetic route that has been developed allows obtaining desired nucleosides without any contamination with unwanted impurities.
- the N- 2- acetyl protected guanosine nucleosides and other N-2 acetyl protected nucleosides having various T- protecting group discussed or T- modification, such as T- fluoro or T- amino grioups can be combined with cyanoethyl phosphate protecting group and utilized in high purity RNA synthesis
- nucleic base in our invention protected with N-2 acetyl guanosine (Acetyl; Ac).
- the solid support having protected nucleosides containing 5'- DMT group and 3' terminus is attached to solid support (Formula 2).
- B a) Adenine (N-bz), b) Cytosine (N-acetyl), c) Guanine (N-acetyl), d) 5-methyl cytosine( N- Fmoc), e) 5- bromocytidine( N-Fmoc), i) 5- iodo cytosine(N- Fmoc), j) 5- fluorocytosine( N-Fmoc), k) 2,6-diaminopurine(N-Fmoc), 1) 2- amino purine( N-Fmoc)
- the nucleosides can be used in oligonucleotide synthesis.
- the base deprotection step can be performed in mild basic conditions or a tertiary amine, secondary amines, such as piperidine capable of removal of Fmoc protecting group via B- elimination in solution phase or on solid support without oligonucleotide detachment.
- kits that contain one or more of the disclosed nucleoside compositions.
- Fig.la HPLC Chromatogram of 5'-DMT-ribo Guanosine (n-acetyl)
- Fig.lb UV Analysis of 5'-DMT -ribo Guanosine (n-acetyl)
- Fig. Ic Table of the UV Analysis of 5'-DMT-ribo Guanosine (n-acetyl)
- Fig.2a HPLC Chromatogram of 5'-DMT-2'-TBDMS-ribo Guanosine (n-acetyl)
- Fig.2b UV Analysis of 5'-DMT -2'-TBDMS-ribo Guanosine (n-acetyl)
- Fig.2c Table of the UV Analysis of 5'-DMT -2'-TBDMS-ribo Guanosine (n-acetyl)
- Fig.3a HPLC Chromatogram of 5'-DMT-2'-TBDMS-ribo Guanosine (n-acetyl)- phosphoramidite
- Fig.3b UV Analysis of 5'-DMT -2'-TBDMS-ribo Guanosine (n-acetyl)-phosphoramidite
- Fig.3c Table of the UV Analysis of 5'-DMT -2'-TBDMS-ribo Guanosine (n-acetyl)- phosphoramidite
- Fig.4a HPLC Chromatogram of purified RNA Sequence
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.4b UV Analysis of purified RNA Sequence (G7799G97GAAAGA79GA9A9AGAGG7). Lot No. CH5-9R-45-01 Note: 1. N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.4c Table of UV Analysis of purified RNA Sequence
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.4d ESI-MS Analysis of purified RNA Sequence
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.4e Synthesis Report of RNA Sequence (G7799G97GAAAGA79GA9A9AGAGG7).
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.5a HPLC Chromatogram of purified RNA Sequence
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.5b UV Analysis of purified RNA Sequence
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.5c Table of the UV Analysis of purified RNA Sequence (7979A777A77A9AAA99A7G99G9A9GG9G7A7G9AGAG).
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.5d ESI-MS Analysis of purified RNA Sequence
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- N-2-acetyl-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig. ⁇ a ESI-MS Analysis of purified RNA Sequence
- N-2-ibu-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- Fig.6b Synthesis Report of RNA Sequence
- N-2-ibu-ribo-Guanosine-2'-TBDMS-3'-CNET Phosphoramidite was utilized in the chimera RNA Synthesis.
- the sequence contains 2'-Fluoro C and 2'-Fluoro-U.
- #7 denotes 2'-Fluoro C base and #9 denote 2'-Fluoro U base.
- This invention relates to synthesis of novel N-2-acetyl protected deoxy and ribo- guanosine, N-acetyl protected modified guanosine, their succinates, phosphoramidites, corresponding solid supports that are suitable for high purity DNA and RNA synthesis.
- Our discovery using N- acetyl as guanine purine base protecting group, which is more base labile protecting group as compared to traditional N-2-isobutyryl guanine, is a novel approach to obtain highest purity oligonucleotides.
- This approach is designed to lead to high purity and very clean oligonucleotide, after efficient removal of the protecting groups and to produce high purity therapeutic grade DNA oligonucleotides, RNA oligonucleotides, diagnostic DNA, diagnostic RNA for microarray platform and high purity oligonucleotides in general.
- the deprotection of N-acetyl protecting groups of the natural deoxy and ribonucleosides and modified deoxy and ribonucleotides occurs during a shorter period of base treatment conditions and removal of acetyl group from guanine moieties under such conditions allows synthesis of various DNA and RNA of highest purity for diagnostics and therapeutic application, RNA synthesis anmd high quality SiRNA in general.
- This approach is further designed to use N- acetyl guanine protecting group on various base sensitive nucleoside such as sugar fluorinated oligo nucleotides synthesis.
- nucleic bases in our invention protected with N-2 acetyl guanine and sugar moiety in the nucleosides carry a 5'- DMT group in deoxyribonucleosides, T- tert butyl dimethyl silyl and 3'-cyanoethylphosphoramidite (CED) ( group 1)
- N-2 acetyl guanine and sugar moiety in the nucleosides carry a 5'- DMT group in deoxyribonucleosides
- 2'- fluoro group and 3'-cyanoethylphosphoramidite (CED) group 2
- N-2 acetyl guanine and sugar moiety in the nucleosides carry a 5'- DMT group in deoxyribonucleosides
- N-2 acetyl guanine and sugar moiety in the nucleosides carry
- N-2 acetyl guanine and sugar moiety in the nucleosides carry a 5'- DMT group in deoxyribonucleosides ( group 5), 5'-DMT-3'-succinyl-Icaa CPG-N-acetyl guanine protected nucleosides (group 6) .5'-DMT-2'-TBDMS -N- acetyl protected- guanosine -3'-amidites
- the invention also contemplates method for preparing the disclosed compositions.
- the products are invariably crystallized.
- RNA oligomers A method for 3' to 5' direction of oligonucleotide bond formations shown in formula 4 in synthetic RNA oligomers.
- the RNA could consist of natural of modified nucleo bases as described in claim 1 to synthesize gapmers, phosphodiesters, phosphorothiates, phosphoselenate.
- One of the nucleoside components will have N- acetyl guanosine base.
- the synthesis could be performed on automated, semi automated DNA/RNA or other synthesizers or manually. The synthesis can be performed at various scales from microgram to kilogram scales.
- RNA oligomers A method for DNA & RNA synthesis using mild amine, secondary or tertiary amine for removal of N-acetyl protecting group of guanosine and cyanoethyl phosphate protecting group for RNA synthesis via 3' to 5' direction of oligonucleotide bond formations shown in formula 4 in synthetic RNA oligomers.
- the RNA could consist of natural of modified nucleo bases, gapmers, phosphodiesters, phosphorothiates, phosphoselenate.
- the nucleoside components will have N- Fmoc as protecting group.
- the synthesis could be performed on automated, semi automated DNA/RNA or other synthesizers or manually. The synthesis can be performed at various scales from microgram to kilogram scales.
- RNA synthesis using mild basic conditions , such as methyl amine solution, alkylamines , secondary or tertiary amine for removal of N-acetyl guanosine protecting group and cyanoethyl phosphate protecting group for RNA synthesis via 3' to 5' direction of oligonucleotide bond formations shown in formula 4 in synthetic RNA oligomers. This is followed by wash of liberated protecting groups.
- the RNA could consist of natural of modified nucleo bases, gapmers, phosphodiesters, phosphorothiates, phosphoselenate.
- the nucleoside components will have N- Fmoc as protecting group.
- the synthesis could be performed on automated, semi automated DNA/RNA or other synthesizers or manually. The synthesis can be performed at various scales from microgram to kilogram scales.
- DNA and RNA synthesis can be carried out via 3' to 5' direction of oligonucleotide bond formations. This is followed by wash of liberated protecting groups.
- the support bound DNA and RNA could consist of natural or modified nucleo bases, gapmers, phosphodiesters, phosphorothiates, phosphoselenate.
- the nucleoside components during such oligo synthesis will have N-acetyl as protecting group.
- the synthesis could be performed on automated, semi automated DNA/RNA or other synthesizers or manually.
- the synthesis can be performed at various scales from microgram to kilogram scales.
- the modified nucleosides incorporated by this method could consists of one or more of purine or pyrimidine modifications such as but not limited to , 5- Fluoro-U, 5-Fluoro dU, 5- fluoro-dC, 5-Fluro-rC, pseudouridine, 5-methyl-dU, 5-methyl-rU, 5-methyl-dC, 5-methyl-rC, 5-bromo-dU, 5-bromo-rU, 5-bromo-dC, 5-bromo-rC, 5-iodo-dU, 5-iodo-rU, 5-vinyl-dU, 5- vinyl-rU, 5-vinyl thymidine, N-3 methyldeoxy uridine, N-3 methyl-ribouridine, N-3 methyl thymidine, 4-thio uridine, 4-thio-2'-deoxyuridine, 2,6-diaminopurine deoxy riboside, N-3 methyl ribothymidine, 2, 6-di
- modified nucleosides could consist of 2'- deoxy-2'-fluoro ribo nucleosides (2'-F-ANAs) such as A, C, G, U , Inosine and modified nucleosides containing T- Fluoro, in one or more positions of an RNA or DNA sequence synthesized by this method.
- 2'-F-ANAs 2'- deoxy-2'-fluoro ribo nucleosides
- modified nucleosides could consist of 2'- deoxy-2'-methoxy ribo nucleosides (2'-OMe-) such as A, C, G, U , Inosine and modified nucleosides containing T- methoxy, in one or more positions of an RNA or DNA sequence synthesized by this method.
- 2'-OMe- 2'- deoxy-2'-methoxy ribo nucleosides
- modified nucleosides could consist of 2'- deoxy-2'-amino ribo nucleosides (2'-NH2) such as A, C, G, U , Inosine and modified nucleosides containing T- amino, in one or more positions of an RNA or DNA sequence synthesized by this method.
- 2'-NH2 2'- deoxy-2'-amino ribo nucleosides
- modified nucleosides could consist of 2'- deoxy-2'-terminal amino ribo nucleosides (2'-terminal NH2), attached via spacer from 2-10 atoms on nucleosides such as A, C, G, U , Inosine and modified nucleosides containing T- terminal amino, in one or — more positions of an RNA or DNA sequence synthesized by this method.
- modified nucleosides could consist of 2'- deoxy-2'-methoxy ethoxy ribo nucleosides (2'-MOE), such as A, C, G, U , Inosine and modified nucleosides containing T- MOE, in one or more positions of an RNA or DNA sequence synthesized by this method.
- 2'-MOE 2'- deoxy-2'-methoxy ethoxy ribo nucleosides
- modified nucleosides could consist of other T- O-alkyl groups, such as T- deoxy-2' -ethoxy, propargyl, butyne ribo nucleosides (2'-OEt, O-Propargyl, 2'-O- Butyne), such as A, C, G, U , Inosine and modified nucleosides containing T- 2'-OEt, O- Propargyl, 2'-0-Butyne, in one or more positions of an RNA or DNA sequence synthesized by this method.
- T- O-alkyl groups such as T- deoxy-2' -ethoxy, propargyl, butyne ribo nucleosides (2'-OEt, O-Propargyl, 2'-O- Butyne), such as A, C, G, U , Inosine and modified nucleosides containing T- 2'-OEt, O- Propargyl, 2'-0-Butyne, in one
- modified nucleosides could consist of 2'- deoxy-2'-fluoro arabino nucleosides (2'-F-ANAs) such as A, C, G, LJ , Inosine and modified nucleosides containing
- modified nucleosides could consist of T- deoxy-2'-fluoro 4'- thioarabino nucleosides (4'-S-FANAs) such as A, C, G, U, Inosine and modified nucleosides containing 4'-S-FANAs in one or more positions of an RNA or DNA sequence synthesized by this method,
- the RNA comprising one or more 2 '-5'- linkage within the sequence , at the 3'- end of the sequence or at the 5'- end of the sequence,
- RNA synthesis comprising T- triisopropylsilyloxy methyl (TOM), protecting group
- RNA synthesis comprising T- X- butyldithiomethyl ( DTM) protecting group
- DTM T- X- butyldithiomethyl
- FANA reverse RNA synthesis comprising the modified base comprising 2'- deoxy-2'- fluoro- beta-D arabinonucleic acid
- RNA synthesis comprising the modified base comprising 4'-thio- T- deoxy-2'- fluoro- beta-D_arabinonucleic acid (4'-Thio- FANA).
- RNA synthesis comprising the modified sugar comprising T- OMethyl modification
- RNA synthesis comprising the modified sugar comprising Bicyclic locked nucleic acids
- RNA synthesis comprising the modified sugar altritol sugar to lead to modified oligonucleotides (ANA),
- ANA modified oligonucleotides
- RNA synthesis comprising the step of conjugation of peptides, such as cell penetrating peptides (CPPs) or membrane permeant peptide (MPPs) utilizing either the free amine function of such peptides and a 3'- terminal carboxylic function on the reverse synthesized
- RNA The CPPs and MPPs having an appropriate carboxyl function can be coupled to the free terminal amino function of an FMOC protected nucleotide or an oligonuleotide.
- the DNA and RNA synthesis comprising the 2'-5'- linked DNA units or 2'-5'- RNA units within the sequence , at the 3'- end of the sequence or at the 5'- end of the sequence.
Abstract
Cette invention concerne une nouvelle méthode de synthèse de l'ARN utilisant la guanine N-2-acétyl protégée comme base nucléosidique, des nucléosides, des succinates, des phosphoramidites, des supports solides correspondants appropriés pour la synthèse d'oligodésoxynucléosides et d'ARN (oligonucléotide). L'utilisation de la guanice N-2-acétyl protégée comme groupe protecteur de base nucléosidique, qui est un groupe protecteur labile de base significativement plus rapide et significativement plus stable que le composé 2-isobutyryl guanosine habituellement utilisé, constitue une nouvelle approche pour obtenir des oligonucléotides de la plus grande pureté. Cette approche est conçue pour produire des oligonucléotides de très grande pureté et très propres, après élimination efficace des groupes protecteurs, notamment le group acétyle de la guanine, et pour produire de l'ADN (oligonucléotide) et de l'ARN (oligonucléotide) thérapeutiques de grande pureté, de l'ADN diagnostique et de l'ARN diagnostique pour puces à ADN. La déprotection des groupes protecteurs acétyle des désoxyribonucléosides et des ribonucléosides naturels se produit en un délai de contact sensiblement réduit dans des conditions de déprotection modérées (bases légères, amines secondaires) car l'élimination de ces groupes dans ces conditions permet la synthèse de différents ADN et ARN de la plus grande pureté pour les applications diagnostique et thérapeutiques. Cette approche est conçue pour obtenir des chimères oligonucléotidiques thérapeutiques de grande pureté à grande échelle qui consistent en la modification d'un fluoro-sucre et de désoxynucléosides, désoxyribonucléosides, nucléosides à base modifiée et à sucre modifié. Cette approche permet également d'utiliser le groupe protecteur acétyle de la guanine quand d'autres bases sont des nucléosides sensibles, et pour supporter des oligonucléotides liés.
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US13/261,029 US9884885B2 (en) | 2009-05-18 | 2010-05-19 | Synthesis of labile base protected-modified deoxy and modified ribo nucleosides, corresponding phosphoramidites and supports and their use in high purity oligonucleotide synthesis |
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US21649109P | 2009-05-18 | 2009-05-18 | |
US61/216,491 | 2009-05-18 |
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PCT/US2010/001494 WO2010134992A2 (fr) | 2009-05-18 | 2010-05-19 | Synthèse de désoxyribonucléosides ou de ribonucléosides modifiés par une base labile protégée, phosphoramidites et supports correspondants et leur utilisation dans la synthèse d'oligonucléotides de grande pureté |
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Cited By (1)
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WO2020020608A1 (fr) | 2018-07-23 | 2020-01-30 | Dna Script | Synthèse enzymatique massivement parallèle de brins d'acides nucléiques |
Citations (4)
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US20030175906A1 (en) * | 2001-07-03 | 2003-09-18 | Muthiah Manoharan | Nuclease resistant chimeric oligonucleotides |
US20060058266A1 (en) * | 2004-08-10 | 2006-03-16 | Muthiah Manoharan | Chemically modified oligonucleotides |
US20060142557A1 (en) * | 1994-03-29 | 2006-06-29 | Sirna Therapeutics, Inc. | 2'-deoxy-2'alkylnucleotide containing nucleic acid |
US20080146787A1 (en) * | 2006-09-02 | 2008-06-19 | Zoltan Timar | Thioether substituted aryl carbonate protecting groups |
Family Cites Families (1)
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CA2176035A1 (fr) * | 1993-11-08 | 1995-05-18 | Nassim Usman | Acide nucleique enzymatique a modification de base |
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- 2010-05-19 WO PCT/US2010/001494 patent/WO2010134992A2/fr active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US20060142557A1 (en) * | 1994-03-29 | 2006-06-29 | Sirna Therapeutics, Inc. | 2'-deoxy-2'alkylnucleotide containing nucleic acid |
US20030175906A1 (en) * | 2001-07-03 | 2003-09-18 | Muthiah Manoharan | Nuclease resistant chimeric oligonucleotides |
US20060058266A1 (en) * | 2004-08-10 | 2006-03-16 | Muthiah Manoharan | Chemically modified oligonucleotides |
US20080146787A1 (en) * | 2006-09-02 | 2008-06-19 | Zoltan Timar | Thioether substituted aryl carbonate protecting groups |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2020020608A1 (fr) | 2018-07-23 | 2020-01-30 | Dna Script | Synthèse enzymatique massivement parallèle de brins d'acides nucléiques |
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US20120065386A1 (en) | 2012-03-15 |
US9884885B2 (en) | 2018-02-06 |
WO2010134992A3 (fr) | 2011-04-07 |
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